The establishment of many novel bioluminescent enzymes (luciferases) has recently been reported. For example, Promega reported the establishment of a novel bioluminescent enzyme originating from a deep-sea shrimp (Non-patent Literature 1). The molecular weight of this enzyme is half (19 kD) that of a known Renilla luciferase (RLuc), and its luminescence intensity is a 100-fold increase. Further, Shigeri et al. reported 11 types of plankton-derived bioluminescent enzyme (Non-patent Literature 20). Some of these bioluminescent enzymes were evaluated as having a luminescence intensity comparable to that of RLuc.
Further, some deep-sea luminescent animals belonging to the Augaptiloidea superfamily have heretofore been discovered (Non-patent Literature 2). In addition, a bioluminescent enzyme originating from Gaussia princeps (GLuc), a bioluminescent enzyme originating from Metridia longa (MLuc), and bioluminescent enzymes originating from Metridia pacifica (MpLuc1 and MpLuc2), which all belong to the Metridinidae family, have also been discovered (Non-patent Literature 15, 19, and 21).
Moreover, InvivoGen has recently established an artificial bioluminescent enzyme, “Lucia,” which is similar to the bioluminescent enzymes originated from copepods (http://www.invivogen.com/lucia).
Meanwhile, research for improving the luminescence intensity or luminescence stability of these bioluminescent enzymes has progressed. Loening et al. established stable RLuc variants having high luminescence intensity by using a method of introducing amino acid mutations into RLuc (Non-patent Literature 14). In their study, a “consensus sequence-driven mutagenesis strategy” was used to specify the mutation introduction site (Non-patent Literature 13). Further, the present inventors also succeeded in improving the luminescence intensity and luminescence stability of GLuc, MpLuc1, and MLuc, which are bioluminescent enzymes originating from deep-sea luminescent animals, by using a method of predicting the enzyme active site based on a hydrophilic amino acid distribution chart, and introducing a variant into the site (Non-patent Literature 11).
The present inventors previously suggested production of a thermodynamically stable bioluminescent enzyme sequence from an attempt to obtain information regarding the luminescence characteristics by bisecting a single bioluminescent enzyme sequence, and aligning the first half and the second half of the sequence based on the homology of amino acids (single sequence alignment; SSA) (Non-patent Literature 3). This method is based on the premise that a marine animal-derived bioluminescent enzyme has two enzyme active sites. By aligning the two split-enzyme active sites based on amino acid similarity, it is possible to easily examine the similarity between the former half and the rear half of the active site. This method attempts to produce a thermodynamically stable bioluminescent enzyme sequence by increasing the similarity of the former and rear half of the sequence on the aforementioned presumption that the amino acid frequency is relevant to the thermodynamic stability (Patent Application No. 2012-237043).
Meanwhile, various applied technologies using a bioluminescent enzyme as a “reporter” have also been developed. Niu et al. classified the bioassays using a bioluminescent enzyme as a reporter into three groups: “basic,” “inducible,” and “activatable” (Non-patent Literature 16). This classification is based on the characteristics of the reporter gene. First of all, the difference between “basic” and “inducible” is the presence or absence of an expression controlling character in the reporter expression by the promoter, and the difference in expression amount. A later-described antibody having a bioluminescent enzyme attached thereto corresponds to “basic,” and the bioluminescence resonance energy transfer (BRET) and two-hybrid assay belong to the category of “inducible.” The reporter-gene probes, which belong to the category of “activatable,” are characterized in that the reporter actively responds to ligand stimulation and produces bioluminescence. The later-described protein complementation assay (PCA), protein splicing assay (PSA), integrated-molecule-format bioluminescent probe, bioluminescent capsule, and the like belong to the category of “activatable.”
For the bioassays (hereinafter may also be simply referred to as “reporter assays”) using these bioluminescent enzymes as a reporter, various bioluminescent probes have been actively developed based on the aforementioned novel bioluminescent enzymes. The present inventors have heretofore conducted research and development regarding bioluminescence imaging using unique molecular design technology. More specifically, the inventors developed a method of measuring translocation of transcription factors into the nucleus or nongenomic protein-protein interactions in the cytosol using protein splicing (Non-patent Literature 7 and 8), and an integrated-molecule-format bioluminescent probe in which all of the necessary elements for signal recognition and bioluminescence emission are integrated (Non-patent Literature 4 and 6). Thereafter, the probes were multicolorized, and developed to be capable of simultaneous imaging of multiple signal-transduction processes (Non-patent Literature 12). Moreover, the inventors further developed a circular permutation technique (Non-patent Literature 9) and a molecular design technology using low-molecular-weight bioluminescent enzymes (Non-patent Literature 12) as strategies for improving the ligand sensitivity of the bioluminescent probe. These technologies have been used as means for efficiently measuring molecular phenomena in cellular and cell-free systems.
Regarding the main research tools for exploring intra- or extracellular molecular phenomena, fluorescence imaging has been used more widely than luminescence imaging. However, due to their autofluorescence property, fluorescent proteins generate a high background, requiring an external light source. Therefore, fluorescence imaging requires a large instrumentation, such as a fluorescence microscope, and a sophisticated light-filtering system. Fluorescence imaging also has a drawback in that the maturation of a fluorescence chromophore takes at least several hours to several days. Further, since the number of simultaneously observable cells is limited for each measurement with a fluorescence microscope, quantitative measurement has been problematic (Non-patent Literature 8).
On the other hand, bioluminescence imaging using a bioluminescent enzyme has, despite its many advantages, a critical problem regarding poor luminescence intensity of bioluminescent enzymes. This problem has decreased the popular use of bioluminescence imaging, compared with fluorescence imaging. Because of this poor bioluminescence intensity of bioluminescent enzymes, high-sensitivity detectors were required; therefore, bioluminescent enzymes have been considered inappropriate for single-cell imaging or exploration of organelles.
Further, studies on multicolor fluorescent proteins have greatly progressed, and many facts regarding their coloring mechanisms have been discovered; thus, many fluorescent proteins with diversified fluorescent characters have been developed based on these study results.
In contrast, only limited kinds of bioluminescent enzymes allow multiple colors. Although it has been known that the diversification of bioluminescent colors is advantageous in that (i) it enables the simultaneous measurement of multiple cellular signals, and that (ii) it ensures a tissue permeability of red-shifted bioluminescence in living subjects, nearly no systematic study for diversifying the colors of bioluminescent enzymes based on their luminescence mechanisms has been conducted.
In addition, appropriate selection of the reaction solution is an important factor in bioassays, and may influence the assay results. In particular, (1) reporter-gene assay, (2) two-hybrid assay, (3) enzyme-linked immunosorbent assay, and (4) radioimmunoassay (RIA) (Non-patent Literature 22 and Non-patent Literature 23) require more careful selection of the reaction solution.
Recently, the present inventors developed a multiple recognition-type bioluminescent probe, which is fabricated by combination of reporter-gene assay and integrated-molecule-format bioluminescent probe (Non-patent Literature 27). This probe is characterized by two sensing steps for a single target substance. The present inventors further developed a multicolor bioluminescence imaging probe set by combining two integrated-molecule-format bioluminescent probes with distinctive colors (Patent Literature 5). This probe set is characterized by multicolor imaging of multiple aspects of bioactivity of a test substance.
Bioassays require a reaction solution, which may be roughly classified into (1) a method using a fluorescent protein and (2) a method using a bioluminescent enzyme (luciferase), depending on the type of the luminescence signal. In the method using a fluorescent protein, a high background is generated due to the autofluorescence, and an external light source is necessary. Further, a relatively large luminescence detector having a precise spectral filter is problematically necessary to measure the fluorescence (e.g., a fluorescence microscope) (Non-patent Literature 8). On the other hand, although the method using a bioluminescent enzyme does not have the above problems, it indispensably requires substrates because of a drawback such that the light emission of bioluminescence is weaker than that of fluorescence. Further, since the method using a bioluminescent enzyme relies on the luminescence of enzyme, easy changes in luminescence quantity depending on the salt concentration, temperature, pH, heavy-metal ion concentration, and the like become problematic. The method using fluorescence also has similar problems. Therefore, to fix the pH and optimize the luminescence reaction conditions, reaction solutions are widely used both in the fluorescence method and the luminescence method.
To improve the assay effect, various additives have been used for reaction solutions (assay buffer). The additives must have functions for ensuring homogenous assay conditions, including (1) prevention of protein decomposition by protease, (2) suppression of influences of interfering substances, (3) ensuring the function as a buffer solution for supporting stable signal generation, and (4) allowing mild lysis of the plasma membrane. Therefore, the additives (5) must stabilize the protein and (6) must not inhibit the probe performance that is the core of the luminescence reaction.
The major additives of the reaction solution include, as salts, NaCl, KCl, (NH4)2SO4, and the like; as an SH reagent, mercaptoethanol, DTT, and the like; as a polyol, glycerol, sucrose, and the like; and as a chelating reagent, EGTA, EDTA, and the like.
Examples of surfactants include polyoxyethylene (10) octylphenyl ether (Triton X-100; TX100), Nonidet P-40 (NP40), polyoxyethylene sorbitan monolaurate (Tween 20; TW20), polyoxyethylene sorbitan monooleate (Tween 80; TW80), polyoxyethylene (20) cetyl ether (Brij58), sodium dodecyl sulfate (SDS), and the like. Heretofore, a suitable surfactant has been selected by referring to the order of the hydrophilic degree of the surfactants, which is TW20>Brij58>TW80>TX100>NP40, and the order of the degree of surface activity, which is NP40>TX100>Brij58>TW20>TW80.
Examples of protease inhibitors to be used for inhibiting protein decomposition include aprotinin (molecular weight: 6.5 kD), leupeptin (molecular weight: 427), pepstatin A (molecular weight: 686), phenylmethylsulfonyl fluoride (PMSF, molecular weight: 174), antipain (molecular weight: 605), chymostatin (molecular weight: 608) and the like. Further, Pefabloc SC (AEBSF, 240 Da), DFP (184 Da), p-APMSF (216 Da), STI (20,100 Da), leupeptin (460 Da), N-tosyl-L-phenylalaninechloromethylketone, 3,4-dichloroisocoumarin (215 Da), EDTA-Na2 (372 Da), EGTA (380 Da), 1,10-phenanthroline (198 Da), phosphoramidon (580 Da), Dithiobis (2-amino-4-methylpentane), E-64 (357 Da), cystatin, bestatin, epibestatin hydrochloride, aprotinin, minocycline, ALLN (384 Da), and the like have been used as protein decomposition inhibitors.
Further, the functional chemical substances below may also be added. By adding sodium molybdate, it is possible to stabilize the receptors and thus protect them from decomposition. Glycerol can be used as a protein preserving agent. Dithiothreitol (DTT) has been used as a reducing agent.
Additionally, as buffers, p-toluenesulfonic acid, tartaric acid, citric acid, phthalate, glycine, trans-aconitic acid, formic acid, 3,3-dimethylglutaric acid, phenylacetic acid, sodium acetate, succinic acid, sodium cacodylate, sodium hydrogen maleate, maleic acid, sodium phosphate, KH2PO4, imidazole, 2,4,6-trimethylpyridine, triethanolamine hydrochloride, sodium 5,5-diethylbarbiturate, N-ethylmorpholine, sodium pyrophosphate, tris(hydroxymethyl)aminomethane, bicine, 2-amino-2-methylpropane-1,3-diol, diethanolamine, potassium p-phenolsulfonate, boric acid, sodium borate, ammonia, glycine, Na2CO3/NaHCO3, sodium borate, or a combination of these substances, have been used.
As described above, the trend of the previous studies has been directed toward the establishment of novel high-intensity bioluminescent enzymes and the enhancement of luminescence stability, heat resistance, or salt resistance. The establishment of bioluminescent enzymes with a red-shifted luminescence spectrum has also attracted attention in recent studies. Not only the establishment of bioluminescent enzymes but also the optimization of the reaction solutions is expected to contribute to improving the stability of luminescence signals, luminescence intensity, and signal-to-noise ratio (S/N ratio). To achieve the object, attempts to optimally prepare various additives (e.g., preservatives, surfactants, and protease inhibitors) have been made.
A substrate, another factor that enables luminescence intensity, luminescence stability, and red-shifted luminescence, finds its basis in studies several decades ago, and the technical progress remains far too slow. The reason for this slow progress is that (1) because studies on luminescent substrates essentially involve organic synthesis, only a limited group of researchers capable of performing organic synthesis have been engaged in studies in this field; and (2) because, among bioluminescent enzymes, marine animal-derived bioluminescent enzymes are particularly susceptible to oxidation in the air, the synthetic environment must be well prepared.
The definition of a substrate is not precisely established and has been changed over time. Luciferin was initially defined as a substance indispensable for the luminescent reaction and an extract from a luminescent organ of animals. In around 1960, the luciferin of Cypridina was discovered from bioluminescent fish parapriacanthus, and during 1790 to 1980, coelenterazine was discovered. Such luciferins were isolated from a variety of luminescent animals and identified as a substrate for many marine animal-derived bioluminescent enzymes (Non-patent Literature 5).
The present invention focuses on coelenterazine as a substrate usable for the luminescent reaction of artificial bioluminescent enzymes and unveils the chemical structure and reaction mechanism of a substrate optimum to artificial bioluminescent enzymes. Coelenterazine was first isolated from Renilla reniformis in the mid-1960s. In 1977, the chemical structure was elucidated by efforts of Inoue et al. (Non-patent Literature 10). Shimomura (2008 Nobel laureate in chemistry) named the substance as coelenterazine in 1975.
Subsequent studies based on these findings on coelenterazine made by the foregoing researchers led to the synthesis of more than 50 coelenterazine derivatives (Non-patent Literature 30 and 31). Coelenterazine derivatives have been studied for more than the past three decades and reported in many non-patent documents. Coelenterazine and its derivatives are thus considered no longer patentable, and recent patent applications in this field are more focused on the development of novel synthesis routes (Patent Literature 6).
Traditionally, coelenterazine and its derivatives have been synthesized through the following synthesis route (Non-patent Literature 17, 18, 24, 26, 28, and 29).

Palladium-catalyzed cross coupling (Stille coupling) was established in relatively recent years as a simpler coelenteramine synthesis technique. The synthesis route follows the procedure described below (Non-patent Literature 32, 33, and 34).

Although many coelenterazine derivatives have been synthesized over the past decades in accordance with the basic synthesis technique, most of the synthesized derivatives have rarely been used because of the unsatisfactory luminescence intensity and luminescence stability. Despite the difficulties involved in the derivative synthesis, native coelenterazine has still been widely used. In addition, not many studies have been made on efficient substrates suitable for bioluminescent enzymes, which is also another reason why this field remains relatively unexplored.
Accordingly, there has been urgent demand for search for efficient substrates that are suitable for bioluminescent enzymes and that have overcome the conventional luminescence intensity and stability problems.